1. Field of the Invention
This invention relates to a novel vaccine against Pasteurella haemolytica which offers superior protection and safety over existing vaccines.
2. Description of the Prior Art
Pasteurella haemolytica is a common respiratory pathogen of animals, particularly bovine, sheep, goats, and exotic zoo ruminants. For example, P. haemolytica serotype 1 (Ph 1) is involved with most acute fibrinohemorrhagic pneumonias that develop in market stressed feeder/stocker calves after shipment (Lillie, 1974, Can. Vet. J., 15:233-242). While development of a safe and effective vaccine would greatly benefit the cattle industry, prevention of pneumonic pasteurellosis has proven to be quite difficult (Frank and Smith, 1983, Am. J. Vet. Res., 44:981-985; Mosier et al., 1989, Am. J. Vet. Res., 47:1-10). A number of vaccines composed of bacteria and viruses have been examined in recent years for the prevention of this disease. However, most of these vaccines have had little positive effect, and bovine pasteurellosis remains a major concern (Martin, 1983, Can. Vet. J., 24:10-19).
The experimental induction of acute bovine respiratory tract lesions in cattle by P. haemolytica alone stimulated a great deal of research examining the use of P. haemolytica bacterins as vaccines. In the 1970s and 1980s, three studies produced an experimental fibrinous pneumonia similar to that seen in actual cases of shipping fever (Friend et al., 1977, Can. J. Compar. Med., 41:219-223; Carter, 1973, J. Amer. Vet. Med. Assoc., 163:863-864; Gibbs et al., 1984, Res. Vet. Sci., 37:154-166). Early results employing bacterin vaccines did not look promising. In a study published by Friend in 1977 (Friend et al., 1977, Can. J. Compar. Med., 41:77-83), killed P. haemolytica given concomitantly by aerosol and subcutaneous routes produced more severe lesions in vaccinated animals than the controls, following intratracheal P. haemolytica challenge. Wilke et al. (1980, Am. J. Vet. Res., 41:1773-1778) observed similar negative effects in studies in which calves were injected subcutaneously with a formalin-killed P. haemolytica vaccine. They observed that protection against P. haemolytica following intrabronchial challenge with this organism was lowered compared with controls. Perhaps the puzzling results obtained with bacterins stimulated the examination of live vaccines for prevention of pneumonic pasteurellosis. Aerosolization or subcutaneous vaccination with live P. haemolytica or P. multocida produced decreased severity of lung lesions induced by transthoracic challenge (Corstvet et al., 1978, Amer. Assoc. Vet. Lab. Diag., 21:67-90; Newman et al., 1982, Am. J. Vet. Res., 43:417-422), as compared to unvaccinated controls. One of the most interesting points about one of those studies (Newman et al.) was that resistance (probably mediated by more efficient phagocytosis by pulmonary macrophages) was greater in aerosol vaccinated than in subcutaneously vaccinated calves. Subsequent studies with goats inoculated in the lung with live P. haemolytica embedded in agar beads yielded a high degree of immunity (Purdy et al., 1990, Am. J. Vet. Res., 51:1629-1634). The vaccinated animals were better protected than controls against a transthoracic challenge of P. haemolytica (1.times.10.sup.7 CFU) injected into the lung of each.
Confer et al. disclosed that vaccination with live P. haemolytica produced an increase in antibody titer to somatic antigens (LPS), leukotoxin (LKT) and a capsular-carbohydrate antigen (CPS) (Lessley, et al., 1985, Vet. Immunol. Immunopathol., 10:279-296). Subsequent reports have shown that elevated serum antibody titers to the P. haemolytica LKT or the CPS correlate well with enhanced resistance to experimental P. haemolytica challenge (Confer et al., 1984, Am. J. Vet. Res., 46:2543-2545, Confer et al., 1985, Am. J. Vet. Res., 46:342-347, Pancier et al., 1984, Am. J. Vet. Res., 45:2538-2542, Gentry et al., 1985, Vet. Immunol. Immunopath., 9:239-250). Confer et al. (1985, Vet. Immunol. Immunopath. 10:265-278) examined serum antibodies to antigens derived from a saline extract of Ph 1 and demonstrated a positive correlation with resistance to experimental bovine pneumonic pasteurellosis to several of the antigens. Their data suggested that antibody to polysaccharide antigens may be important to resistance. This led to the incorporation of several of these antigens, LPS, CPS, and LKT, in a subunit agar-bead vaccine (Purdy et al., 1993, Amer. J. Vet. Res., 54:1637-1647). LKT, a heat-labile protein toxin produced by P. haemolytica, has been considered a virulence factor for the organism because of its ability to exert a negative effect on bovine alveolar macrophages and neutrophils (Wilkie, 1982, J. Am. Vet. Med. Assoc., 181:1074-1079). LPS produced by P. haemolytica may be important in bovine pulmonary pasteurellosis. Confer and Simon, (1986, Am. J. Vet. Res., 47:154-157), recently demonstrated biological activity of P. haemolytica LPS for bovine leukocytes in vitro but declared that further studies are required to decide whether it plays a role in the production of the lung lesions seen in pneumonic pasteurellosis. Slocombe et al. (1990, Am. J. Vet. Res., 51:433-438) demonstrated that Ph 1 endotoxin is pathogenic when delivered intravenously (IV) and by airway routes. They showed that intratracheal inoculation of Ph 1 LPS caused hypoxemia and increased the alveolar-arterial oxygen differences. By contrast, IV inoculation of Ph 1 and LPS caused systemic hypotension, leukopenia, and gas exchange impairment. Both routes of inoculation were associated with areas of pulmonary hemorrhage, edema and acute inflammation. In addition, Breider et al. (1990, Infect. Immun., 58:1671-1677) showed that P. haemolytica produces a soluble factor that appears to be LPS and is directly toxic to bovine pulmonary endothelial cells. However, Confer et al. (1986, Amer. J. Vet. Res., 47:1134-1138) examined serum antibodies to P. haemolytica LPS and their relationship to experimental bovine pneumonic pasteurellosis, and they concluded that serum antibody responses to Ph 1 LPS did not seem important for resistance to challenge. They showed no significant correlation between the lung lesion score and antibody response to Ph 1 LPS. This is essentially the same conclusion that was reached by Purdy et al. (1993, ibid), regarding the protective effect of antibodies directed against Ph A1 LPS.
The importance of the CPS in infections caused by P. haemolytica has been known for a long time. The presence of soluble capsular material on P. haemolytica was first demonstrated in 1956 (Carter, 1956, Can. J. Microbiol., 2:485-488) and it was shown to be carbohydrate. Later, capsular material was extracted from P. haemolytica by a variety of different techniques (Evans and Wells, 1979, Res. Vet. Sci., 27:213-217; Gentry et al., 1982, Am. J. Vet. Res., 43:2070-2073; Tadayan and Lauerman, 1981, Vet. Microbiol., 6:85-93) and vaccination of mice, hamsters, and sheep (Wells et al., 1979, Res. Vet. Sci., 27:248-250, Gilmore et al., 1979, Vet. Rec., 104:15) with these extracts protected against experimental challenge with this organism. However, subsequent studies (Purdy et al., 1993, ibid; Purdy et al., 1991, Abstracts, Conference of Research Workers in Animal Disease, #113, p. 20; Conlon et al., 1991, Infect. Immun., 59:587-591; Conlon and Shewen, 1991, Abstracts, Conference of Research Workers in Animal Disease, #277, p. 49) have indicated that a subunit vaccine would not provide adequate protection against a substantial Ph 1 challenge. Transthoracic immunization with Ph 1 LPS and recombinant cytotoxin offered no protection against a subsequent transthoracic challenge. Initial experiments with Ph 1 CPS demonstrated some protection against a subsequent Ph 1 challenge but it was not significant. When these studies were repeated (Purdy et al., 1993, ibid; Purdy et al. 1991, ibid) no protective effect was exhibited. Others have had a similar lack of success with subunit vaccines. Conlon et al. (ibid) decided in their study with recombinant leukotoxin from Ph 1 and cattle that, "although LKT is an important virulence factor for the organisms, an immune response to LKT alone does not protect animals against disease". These same workers (Conlon and Shewen, ibid) also showed that a purified Ph 1-CPS vaccine offered no protection against a subsequent Ph 1 challenge in cattle.
Lo and MacDonald (1991, Mutation Res., 263:159-163) demonstrated that P. haemolytica is highly sensitive to ultraviolet radiation, and suggested that the bacterium lacks some of the important mechanisms to repair UV-induced damage. Whitely et al. (1991, Vet. Pathol., 28:275-285) examined alterations in pulmonary morphology and peripheral coagulation profiles caused by intratracheal inoculation of live and ultraviolet light-killed Ph 1 in cattle. These authors showed that UV-killed bacteria were capable of causing fibrin exudation, platelet aggregation, and alveolar epithelial damage similar to live bacteria, but the degenerative changes in neutrophils, endothelial cells and intravascular fibrin formation that were observed with the live Ph 1, were not seen.